Method for isolating and purifying nucleic acids

ABSTRACT

A method for isolating and purifying nucleic acids with an improved recovery yield is provided. A mixed solution containing the nucleic acids, salts, and an organic solvent is contacted with an adsorption support to cause the nucleic acids to be adsorbed on the support. Then, the nucleic acids are desorbed from the support using an elution buffer. At least one compound containing 2 to 10 carbon atoms as selected from the group consisting of aliphatic ether, aliphatic ester, and aliphatic ketone is used as the organic solvent. The method improves the yield of nucleic acids collection, is easy to implement and less susceptible to contamination.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for isolating and purifyingnucleic acids by eluting nucleic acids from nucleic acid-containingsamples, such as biological materials.

2. Description of Related Art

An efficient method known in the art for isolating and purifying nucleicacids is based on the adsorption of the nucleic acids on glass or silicaparticles in the presence of a chaotropic salt, followed by recoveringthe nucleic acids (Vogelstein, B. and Gillespie, D. (1979); “Preparativeand analytical purification of DNA from agarose”, Proc. Natl. Acad. Sci.USA 76: 615-619). According to this method and using high concentrationsof a chaotropic salt, such as sodium iodide, sodium perchlorate orguanidine thiocyanate, DNA can be isolated and purified from agarose, orRNA or DNA can be isolated and purified from various mixtures (Boom, R.(1990); Rapid and simple method for purification of nucleic acids, J.Clin. Microbiol. 28: 495-503).

Nucleic acids after purification are often subjected to the polymerasechain reaction (PCR). The technique of PCR amplifies nucleic acids in asequence-specific manner and therefore is widely used in genetic or DNAdiagnosis. In utilizing this PCR technique routinely for clinicalpurposes, several problems arise. It is known, among others, thatinhibitor substances remaining un-removed in the nucleic acidspurification step inhibit the PCR. Such inhibitor substances includehemoglobin and surfactants used in the nucleic acids extraction process,for instance. With such a background, it is pointed out that theprocesses for extracting and purifying nucleic acids are important(Oshima et al., JJCL A, 22(2), 145-150 (1997)).

The procedures in the extracting process which have so far been carriedout manually are complicated and requires skills. As such, automation byinstruments is desired. Thus, it is demanded that an extraction methodsuitable for automation be developed, inclusive of reagents to be used.There is a nucleic acids extraction method suitable for automationdescribed in JP-A-127854/1999, and another method using the apparatusdescribed in JP-A-266864/1999. Further, there are reagents allegedlysuitable for nucleic acids extraction on an automated apparatusdescribed in Tokuhyo (Japanese Translation of Unexamined PCT Appln.) No.501321/1996. According to the method using the reagents described in theTokuhyo No. 501321/1996, the nucleic acids to be isolated and purifiedfrom a solution containing a high concentration (ionic strength) ofsalts and a high concentration of alcohol are brought into contact withan adsorption support within a column for genome extraction so as toadsorb them on the support then desorb them from the adsorption supportby means of a solution containing lower concentration (ionic strength)of salts.

However, the method for isolating and purifying nucleic acids using thereagents described in the Tokuhyo No. 501321/1996 has a problem in thatthe yield of nucleic acids collection is low. The method, which uses thecolumn for genome extraction described in JP-A-127854/1999, requires along period of time for contacting the nucleic acids with the adsorptionsupport to adsorb them thereon, since the viscosity of the solution ishigh.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forisolating and purifying nucleic acids by which the recovery yield isimproved and the time required for the nucleic acids recovery is reducedsuch that the method is excellent in operability and is less susceptibleto contamination.

The method for isolating and purifying nucleic acids as provided by thepresent invention comprises allowing nucleic acids to be adsorbed on aninorganic or organic support from a mixed solution comprising a nucleicacid-containing sample, high concentration of salts and an organicsolvent, and then desorbing the nucleic acids from the support with anelution buffer consisting of an aqueous solution. The support is washedwith a washing buffer prior to recovery to remove components other thanthe nucleic acids. The nucleic acids are, for example, derived fromwhole blood.

Chaotropic salts may be used as the salts. In particular, guanidinehydrochloride, guanidine thiocyanate or potassium iodide are used withinthe concentration range of 1 M to 8 M.

The organic solvent (organic compound) comprises at least one compoundcontaining 2 to 10 carbon atoms selected from the group consisting ofaliphatic ethers, aliphatic esters, and aliphatic ketones. Theconcentration of the organic solvents in the mixed solution is 5% to 50%by volume.

As typical example of the aliphatic ether, which can be used as theorganic solvent (organic compound) in the mixed solution, includes atleast one of ethylene glycol dimethyl ether, ethylene glycol diethylether, propylene glycol dimethyl ether, propylene glycol diethyl ether,diethylene glycol dimethyl ether, diethylene glycol diethyl ether,tetrahydrofuran, and 1,4-dioxane.

As typical example of the aliphatic ester used as the organic solvent(organic compound) in the mixed solution comprises at least one ofpropylene glycol monomethyl ether acetate, and ethyl lactate.

As typical example of the aliphatic ketone used as the organic solvent(organic compound) in the mixed solution comprises at least one ofhydroxyacetone, acetone, and methyl ethyl ketone.

In applying the present invention, it is recommended that the mixedsolution contains 0.1% to 50% by volume of a surfactant and 0.1% to 5%by weight of a defoaming agent.

As the inorganic support, a porous or nonporous material consisting ofsilica, alumina, zeolite, titanium dioxide and/or the like is used.

The washing buffer contains a high concentration of organic solventssuch as alcohols, and serves to remove components other than the nucleicacids by repeating a separation procedure of sucking or centrifugation anumber of times.

According to the method for isolating and purifying nucleic acids inaccordance with the present invention, the obtained nucleicacid-containing solution shows a reduced viscosity, the formation ofbubbles can be controlled, and defoaming is promoted, hence theoperability is improved. The yield of nucleic acids recovered orcollected also increases without incurring move contamination.Additionally, the nucleic acids recovery time is shortened.

Other and further objects, features and advantages of the invention willappear more fully from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

The preferred embodiments of the present invention are illustrated inthe accompanying drawings in which:

FIG. 1 shows the nucleic acids extraction process in one embodiment ofthe present invention;

FIG. 2 shows the constitution of whole blood in the embodiments of theinvention;

FIG. 3 shows the lysis process for nucleic extracting the acids from thewhole blood in FIG. 2;

FIG. 4 shows the procedure (first method) for extracting the nucleicacids from the whole blood in the first embodiment of the invention;

FIG. 5 shows the procedure (second method) for extracting the nucleicacids from the whole blood in the second embodiment of the invention;

FIG. 6 shows the correlation between the Diglyme concentration in themixed solution and the respective nucleic acids yield as found in someembodiments of the invention;

FIG. 7 shows the correlation between the EL concentration in the mixedsolution and the respective nucleic acids yield as found in someembodiments of the invention;

FIG. 8 shows the proportion of Diglyme in the additive solutioncomprising Diglyme and EL and the respective nucleic acids yield asfound in some embodiments of the invention;

FIG. 9 shows the correlation between the content of Tween 20 in thelysis buffer and the respective nucleic acids yield as found in someembodiments of the invention;

FIG. 10 shows the correlation between the content of Tween 80 and therespective nucleic acids yield as found in some embodiments of theinvention;

FIG. 11 shows the correlation between the content of Triton X-100 in thelysis buffer and the respective nucleic acids yield as found in someembodiments of the invention; and

FIG. 12 shows the correlation between the level of addition of thedefoaming agent CE-457 to the lysis buffer and the respective nucleicacids yield as found in some embodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present invention, the isolation and purification ofthe nucleic acids from an organism, i.e. DNAs, may be carried out basedon the method illustrated in FIG. 1. FIG. 1 shows the main elements inthe processes, described in JP-A-127854/1999. Following explain thedetails of each process.

FIGS. 2 to 5 illustrate the processes for extracting the nucleic acidsfrom whole blood using the nucleic acids extraction reagents accordingto the processes described in FIG. 1. In the following description,nucleic acids extraction from 100 μL of human whole blood is taken as anexample. As shown in FIG. 2, human whole blood 1 contains erythrocytes2, leukocytes 3, and other components. Nucleic acids are mainly derivedfrom the nucleus 4 of the leukocyte 3.

As shown in FIG. 3, 10 μL of proteinase K (6) is added to a tube 5.Then, 100 μL of whole blood 1 is added to the tube 5. This step isindicated by 101. Then, 100 μL of a lysis buffer is added, and theresulted mixture is stirred. This step is indicated by 102. This lysisbuffer contains 1 M to 8 M of a chaotropic salt and a surfactant at notmore than 50% by volume. The solution in the tube 5 is incubated at 56°C. for 10 minutes, whereby the leukocytes 3 are decomposed, and thenucleic acids within the nucleus come out therefrom into the solution.This step is indicated by 103. Thereafter, 100 μL of an additivesolution (e.g. organic solvent such as EGDEE (ethylene glycol diethylether) or Diglyme (diethylene glycol dimethyl ether)) is added, then thewhole mixture is stirred to provide a mixed solution 7. This step isindicated by 104. The procedure until the step 104 is referred to as a“lysis process”.

After this lysis process, there are several methods available forrecovering or collecting the nucleic acids. Each method comprises anadsorption process, a washing process, and an elution process. Astypical examples, there are a spin column method and a bi-directionalmethod.

FIG. 4 illustrates how the spin column method (hereinafter referred toas “first method”) is applied in the invention. After the additivesolution is added, the mixed solution 7 is transferred from the tube 5to a spin column 8. The inside of the bottom portion of the spin column8 is packed with an adsorption support 9 made of very fine silica orglass wool in a filter paper-like form. Alternatively, the support ismade of organic hybrid materials. The mixed solution 7 containing theadditive solution is passed through the adsorption support 9 by asucking force to cause the nucleic acids to be adsorbed on theadsorption support 9 (adsorption process). This process is indicated by105. Thereafter, 500 μL of a washing buffer 11 containing 20% to 80% ofan organic solvent, such as an alcohol, is poured into the spin column8. This step is indicated by 106. Then, the adsorption support 9 iswashed by a sucking force to remove other components than the nucleicacids. This is step 107 (washing process). This washing process isrepeated twice, but the nucleic acids adsorbed are not desorbed.Thereafter, a low salt concentration elution buffer 12 is added, thenthe mixture is allowed to stand for 2 minutes to 5 minutes. This step isindicated by 108. The nucleic acids are eluted from the adsorptionsupport 9 and finally recovered/collected in a tube 10 by a suckingforce (step 109) (elution process). A typical elution buffer has thefollowing composition: 50 mmol Tris/HCl (pH 8.5) and 0.1 mmol EDTA•2Na.In the first method, a centrifugation procedure can be employed in eachstep for simple and rapid treatment.

FIG. 5 illustrates how the bi-directional method (hereinafter referredto as “second method”) is applied in the invention. In this secondembodiment of the invention, a cylindrical hollow column 14 is used,which has an adsorption support 13 of a lower density of glass or quartzwool or the like than the one in the first embodiment. In this column14, the mixed solution 7 (after adding the additive solution) is passedthrough the support 13 (bi-directionally) a plurality of times (suctionand discharge) to enhance the nucleic acids to be adsorbed on theadsorption support 13. This is step 110 (adsorption process). Thenanother tube 16 containing 500 μL of a washing buffer 15 (containing anorganic solvent, such as an alcohol), is used to remove other componentsthan the nucleic acids. This washing process is carried out at leasttwice. Thereafter, an elution buffer 18 (100 μL) placed in a tube 17 andheated in advance to 70° C. is sucked up to a level such that it comesinto contact with the adsorption support 13 within the column 14. Theelution buffer is allowed to stand in that condition for about 2minutes. This process is indicated by 111. By this procedure, thenucleic acids are eluted from the adsorption support 13 into the elutionbuffer 18. The elution buffer (eluate) 18 is discharged into the tube17, and the nucleic acids are collected. This process is indicated by112. A typical elution buffer has the following composition: 50 mmolTris/HCl (pH 8.5) and 0.1 mmol EDTA•Na.

The materials, reagents and samples mentioned above are described belowin detail.

-   1. Materials for nucleic acids adsorption and columns-   1.1 Spin column

Three layers of Whatman glass fiber filter paper, for instance, arefixed on a column for centrifugal chromatography (spin column). Thecolumn is used according to the first method as shown in FIG. 4.

-   1.2 Column for nucleic acids adsorption

A column has a support made form the same quartz wool with a diameter of0.5 μm to 30 μm of a lower density than the support in the first method(product of Tosoh Quartz Corp. or Toshiba Ceramics Co., Ltd., and usedfor nucleic acids adsorption in JP-A-266864/1999). This is usedaccording to the second method shown in FIG. 5.

-   2. Nucleic acid-containing samples-   2.1 Blood (human)

Blood just collected or frozen at −20° C. is used.

-   2.2 Tissues

Fresh or frozen tissues are used.

-   2.3 Plant

A plant mashed in a mortar is used fresh or frozen in liquid nitrogen.

-   2.4 Cells

Isolated cells or isolated/incubated cells are fresh or in a frozenstate.

-   3. Reagents-   3.1 Lysis buffers

Each lysis buffer comprises chaotropic salts, a surfactants, a defoamingagent, and another salt components, as follow.

-   3.1.1 Chaotropic salts

One of the following is used as the chaotropic salt.

-   -   KI: Potassium iodide    -   GuHCl: Guanidine hydrochloride    -   GuHSCN: Guanidine thiocyanate

-   3.1.2 Surfactants

One of the following is used as the surfactant.

-   -   Tween 20: Polyoxyethylene(20)sorbitan monolaurate    -   Tween 40: Polyoxyethylene(20)sorbitan monopalmitate    -   Tween 60: Polyoxyethylene(20)sorbitan monostearate    -   Tween 80: Polyoxyethylene(20)sorbitan monooleate    -   Tween 85: Polyoxyethylene(20)sorbitan trioleate    -   Triton X-100: Polyoxyethylne(10) isooctylphenyl ether    -   Defoaming agent    -   CE-457: Disfoam, defoaming agent produced by NOFCO (NOF Corp.),        a polyalkylene glycol derivative

-   3.1.4 Other salts

EDTA•2Na: Ethylenediaminetetraacetic acid disodium salt

Tris: Tris(hydroxymethyl)aminomethane

-   3.2 Additive solutions

One of the following organic solvents is used as the additive solution.

-   -   EGDME: Ethylene glycol dimethyl ether    -   EGDEE: Ethylene glycol diethyl ether    -   PGDME: Propylene glycol dimethyl ether    -   PGDEE: Propylene glycol diethyl ether    -   DIGLYME: Diethylene glycol dimethyl ether    -   DGDEE: Diethylene glycol diethyl ether    -   THF: Tetrahydrofuran    -   DX: 1,4-Dioxane    -   PGMEA: Propylene glycol monomethyl ether acetate    -   EL: Ethyl lactate    -   HAC: Hydroxyacetone    -   AC: Acetone    -   MEK: Methyl ethyl ketone

-   3.3 Washing buffers

One of the following compositions is used as the washing buffer.

-   -   W1: 25 mmol/l potassium acetate, 70 volume % ethanol    -   W2: 25 mmol/l potassium acetate, 50 volume % ethanol    -   W3: 25 mmol/l Tris/HCl, 50 volume % ethanol    -   W4: 10 mmol/l Tris/HCl (pH 5), 0.1 mmol/l EDTA•2Na, 50 volume %        ethanol

-   3.4 Elution buffers

One of the following compositions is used as the elution buffer.

-   -   E1: Water (100%), pH 8.0    -   E2: 10 mmol/l Tris/HCl (pH 8.5), 0.1 mmol/l EDTA•2Na    -   E3: 50 mmol/l Tris/HCl (pH 8.5), 0.1 mmol/l EDTA•2Na

-   3.5 Enzymes

One of the following enzymes is used.

-   -   protease: Proteolytic enzyme    -   proteinase K: Alkaline proteinase K

EXAMPLES Example 1 Nucleic Acids Extraction from Whole Blood (The SecondMethod)

According to the second method illustrated in FIG. 3 and FIG. 5, nucleicacids are extracted from the human whole blood 1 (100 μL). In the lysisprocess, the whole blood 1 (100 μL) and a lysis buffer (100 μL) areadded to a tube 5 containing proteinase K (6) (10 μL), followed bystirring into a mixed solution. The lysis buffer used here containsguanidine hydrochloride at a concentration of 3 M and Triton X-100 at 5%by volume. The mixed solution is incubated at 70° C. for 10 minutes.Thereafter, an additive solution (100 μL) selected from among EGDME,EGDEE, PGDME, PGDEE, DIGLYME, DGDEE, THF, DX, PGMEA, EL, HAC, AC, andMEK is added, and the resulted mixture is stirred.

In the adsorption process, the procedure for bi-directional transfer ofthe mixed solution by suction and discharge is repeated 10 times using acolumn 14 packed with an adsorption support 13 (product of ToshibaCeramics Co., Ltd.).

In the washing process, the washing buffer (W1) 15 placed in anothertube 16 undergoes the procedure for bi-directional transfer for threetimes.

In the elution process, an elution buffer 18 placed in a further tube 17is sucked up to a level such that the adsorption support is whollyimmersed in that buffer, and the buffer stands in that condition for 2minutes. Thereafter, the elution buffer is removed by centrifugation.The nucleic acids are thus collected.

The nucleic acids solution obtained could be used in subsequentreactions, such as PCR, or analysis without any purification, such asprecipitation.

Table 1 shows the nucleic acids yields obtained by using variousadditive solutions and the A260/A280 values indicative of the puritiesof the nucleic acids in the respective extracted nucleic acidssolutions.

TABLE 1 No. Additive solution Nucleic acids yield (μg) A260/A280  1EGDME 1.6 1.7  2 EGDEE 1.8 1.8  3 PGDME 1.5 1.7  4 PGDEE 1.4 1.8  5DIGLYME 1.9 1.9  6 DGDEE 1.8 1.8  7 TIIF 1.5 1.7  8 DX 1.5 1.8  9 PGMEA1.4 1.7 10 EL 2.0 1.9 11 IIAC 1.6 1.8 12 AC 1.5 1.7 13 MEK 1.3 1.7

In the above, A260 denotes the absorbance at the wavelength 260 nm ofthe nucleic acid-containing aqueous solution. When the A260/A280 valueis 1.8, the nucleic acids concerned are regarded as being pure. AnA260/A280 value within the range of 1.7 to 1.9 is regarded as beingsufficiently pure. The same enzyme (proteinase K), lysis buffer, washingbuffer, and elution buffer are used in the series of experiments, andthe results are shown in Table 1. The nucleic acids obtained arequantified by fluorometry using the fluorescent dye PicoGreen (MolecularProbes Inc.) for quantifying double-stranded nucleic acids. For eachadditive solution, it appears that high-purity nucleic acids areobtained.

FIG. 6 shows the change in nucleic acids yield along with theconcentration change of the additive solution of Diglyme. The abscissadenotes the Diglyme concentration (% by volume) in the mixed solution 7,and the ordinate denotes the relative yield (%) with the maximum yieldtaken as 100. The relative yield should not be lower than 80% (thedesired yield). In the Diglyme concentration range of 5% to 50%, therelative yields are higher than 80%. In particular, it is seen that themaximum yield is obtained when the concentration is about 43%.

FIG. 7 shows the change in nucleic acids yield along with theconcentration change of the additive solution of EL. In the series ofexperiments, the same enzyme (proteinase K), lysis buffer, washingbuffer, and elution buffer are used. When the relative yield is notlower than 80%, the desired yield is obtained. In the EL concentrationrange of 10% to 50%, the relative yields are higher than 80%. Inparticular, it is seen that the maximum yield is obtained when theaddition level is about 33%.

FIG. 8 shows the change in nucleic acids yield when Diglyme and EL aremixed as the additive solution. Here, the level of addition of theadditive solution corresponds to 33% of the whole mixed solution. InFIG. 8, the abscissa denotes the proportion (by volume) of Diglyme inthe additive solution. When the relative yield is not lower than 80%,the desired yield is obtained. It is seen that the desired yield can beobtained at any proportion of Diglyme.

FIG. 9, FIG. 10, and FIG. 11 each shows the relation between thesurfactant concentration in the lysis buffer and the nucleic acidsyield. In the series of experiments, the results are shown in FIG. 9 toFIG. 11, while the same enzyme (proteinase K), additive solution,washing buffer, and elution buffer are used. When the relative yield isnot lower than 80%, the desired yield is obtained. It is indicated thatrelative nucleic acids yields are higher than 80% when the surfactantcontent is 5% to 50%.

FIG. 12 shows the relation between the level of addition of a defoamingagent (CE-457) in the mixed solution 7 and the nucleic acids yield. Inthe series of experiments, the same enzyme (proteinase K), additivesolution, washing buffer, and elution buffer are used. The lysis buffershave the same composition except for the concentration of the defoamingagent. It is indicated that the nucleic acids yields are higher than 80%within the defoaming agent addition level range of 0.2% to 2.5%. It isthus shown that the addition of a defoaming agent is effective not onlyin preventing the mixed solution from foaming but also in improving thenucleic acids yield.

Table 2 shows that the employment of Diglyme in the additive solutionenhances the adsorption operability (Viscosity coefficient: OrganicSolvents, Fourth Edition, John Wiley & Sons, Inc., 1986).

TABLE 2 Viscosity coefficient at Time required for Additive solution 25°C., η(kg m⁻¹ s⁻¹) adsorption process (s) Ethanol 1.0826 60 Diglyme0.989  50

As compared with ethanol, namely a compound representative of thealcohols described in Tokuhyo No. 501321/1996, the additive solutionDiglyme applied in the present invention is lower in viscosity and,therefore, the viscosity of the mixed solution in the adsorption processbecomes lower, hence the resistance against passage thereof through thecolumn 14 becomes lower, as evidenced by the result that the timerequired for adsorption process is shortened by 10 seconds. Foaming isalso prevented, hence the degree of contamination decreased, and theadsorption operability improved.

Table 3 shows the flash points of various additive solutions (solvents).These solvents are examined for safety based on these data (“YozaiPokettobukku (Solvent Pocketbook)”, edited by the Society of OrganicSynthetic Chemistry, Japan, published by Ohmsha, Ltd.).

TABLE 3 Additive solution Flash point (° C.) Ethanol 13 Diglyme 56 EL 52EGDEE 35 DGDEE 82 EGDME −2 THF −14 DX 12 AC −15 MEK −7

Flash point comparison reveals that the flash points of Diglyme, EL,EGDEE, and DGDEE are higher than the flash point of ethanol. Therefore,the use of Diglyme, EL, EGDEE, and DGDEE, among others, as additivesolutions is less dangerous from the viewpoint of explosion and fire,among other, hence preferred from the safety viewpoint.

Example 2 Nucleic Acids Extraction from Whole Blood (The Second Method)

Nucleic acids are extracted from human whole blood 1 (1 mL) according tothe second method depicted in FIG. 3 and FIG. 5. In the lysis process,the whole blood 1 (1 mL) and a lysis buffer (1 mL) are added to a tube 5containing proteinase K (6) (100 μL), followed by stirring into a mixedsolution. The lysis buffer used here contained a concentration of 3 M ofguanidine hydrochloride and Triton X-100 at 5% by volume. The mixedsolution is incubated at 70° C. for 10 minutes. Thereafter, an additivesolution (1 mL) selected from Diglyme and ethanol is added, and themixture is stirred.

In the adsorption process, the procedure for bi-directional transfer ofthe mixed solution 7 is repeated 10 times using a column 14 packed withan adsorption support 13 (100 mg) (product of Tosoh Quartz Corp.).

In the washing process, the washing buffer 15 (W1) (3 mL) placed inanother tube 16 undergoes the suction/discharge operation forbi-directional transfer for three times.

In the elution process, an elution buffer 18 (1 mL) placed in a furthertube 17 is sucked up to a level such that the adsorption support 13 iswholly immersed in the buffer. The buffer stands in that state for 2minutes. Thereafter, the elution buffer 18 is discharged into the tube17. A nucleic acids solution is thus recovered.

The nucleic acids solution obtained could be used in subsequentreactions and analysis, for example PCR, without any furtherpurification, such as precipitation.

Table 4 shows the nucleic acids yields and the A260/A280 values for thenucleic acids solutions obtained when ethanol and Diglyme arerespectively used as additive solutions.

TABLE 4 Additive solution Nucleic acids yield (μg) A260/A280 Diglyme15.0 1.8 Ethanol  0.6 1.8

While when ethanol is used as the additive solution, the nucleic acidsyield is 0.6 μg, the use of Diglyme gives 15.0 μg. Therefore, Diglyme issuperior additive to the additive solution than Ethanol.

Example 3 Nucleic Acids Extraction from Whole Blood (The First Method)

Nucleic acids are extracted from 100 μL of human whole blood accordingto the first method illustrated in FIG. 3 and FIG. 4.

The lysis process is carried out in the same manner as in Example 1. Thesame enzyme, lysis buffer, additive solution, washing buffer, andelution buffer as in Example 1 are used.

In the adsorption process, the mixed solution is poured into a spincolumn 8 and centrifuged (number of revolutions 6,000 rpm) for 1 minuteusing a desk centrifuge (number of revolutions 6,000 rpm).

In the washing process, a washing buffer (W2) 11 (500 μL) is poured intothe spin column 8, and centrifugation is conducted for 1 minute (numberof revolutions 6,000 rpm).

In the elution process, an elution buffer (E2) 12 (100 μL) is pouredinto the spin column 8. After standing for 2 minutes at room temperature(25° C.), the nucleic acids are collected by 1 minute of centrifugation(number of revolutions 6,000 rpm). The nucleic acids are quantified, andthe purity of the nucleic acids is evaluated.

In this method, the same results as in Example 1 are obtained.

Example 4 Nucleic Acids Extraction from Tissue (The First Method)

Rat liver tissues (25 mg) are mixed with 80 μL of PBS (phosphatebuffered saline) and mechanically homogenized. Protease K (20 μL) isadded, and the mixture is stirred and heated at 56° C. until the tissueis dissolved. A lysis buffer (200 μL) containing a concentration of 3 Mof guanidine hydrochloride and Tween 80 at 5% by volume is added, andthe resulted mixed solution is stirred and heated at 70° C. for 10minutes. Diglyme (200 μL) is added, followed by stirring. The abovesteps constituted the lysis process.

In the adsorption process, the mixed solution is poured into a spincolumn 8 and centrifuged (number of revolutions 6,000 rpm) for 1 minuteusing a desk centrifuge.

In the washing process, a washing buffer (W3) 11 (500 μL) is poured intothe spin column 8, and centrifugation is carried out for 1 minute(number of revolutions 6,000 rpm).

In the elution process, an elution buffer 12 (100 μL) selected from theabove-mentioned E1, E2 and E3 is poured into the spin column 8. Afterstanding for 2 minutes at room temperature (25° C.), the nucleic acidsare recovered by 1 minute of centrifugation (number of revolutions 6,000rpm) such that they are quantified, and the purity of the nucleic acidsis evaluated.

As a result, high-purity nucleic acids with an A260/A280 ratio of 1.8are obtained, and they could be used in the PCR.

Example 5 Nucleic Acids Extraction from Tissue (The First Method)

Nucleic acids are extracted from 20 mg of bladder tissues (rat) in thesame manner as in Example 4. As a result, highly pure nucleic acids withan A260/A280 ratio of 1.7 are obtained. They could be used in the PCR.

Example 6 Nucleic Acids Extraction from Urine (The First Method)

A urine sample (20 mL) is centrifuged (number of revolutions 14,000 rpm)for 5 minutes. The supernatant is removed, and the urine sediments areseparated. The urine sediments is added with 200 μL of a regent solutioncontaining a concentration of 3 M of guanidine hydrochloride and TritonX-100 at 5% by volume. After stirring, the mixture is heated at 70° C.for 10 minutes. Diglyme (200 μL) is added, followed by stirring. Theabove steps constituted the lysis process.

In the adsorption process, the mixed solution is poured into a spincolumn 8 and centrifuged (number of revolutions 6,000 rpm) for 1 minuteusing a desk centrifuge.

In the washing process, a washing buffer (W4) 11 (500 μL) is poured intothe spin column 8, and centrifugation (number of revolutions 6,000 rpm)is effected for 1 minute.

In the elution process, an elution buffer (E3) 12 (100 μL) is pouredinto the spin column 8. After standing for 2 minutes at room temperature(25° C.), the nucleic acids are collected by 1 minute of centrifugationsuch that they are quantified, and the impurity of the nucleic acids isevaluated.

As a result, high-purity nucleic acids with an A260/A280 ratio of 1.8are obtained, and they could be used in the PCR.

Example 7 Nucleic Acids Extraction from Cells (The First Method)

10⁶ HeLa cells are dispersed in PBS (100 μL), 200 μL of a lysis buffercontaining a concentration of 3 M of guanidine hydrochloride and TritonX-100 at 5% by weight is added, and the mixture is stirred and heated at70° C. for 10 minutes. Diglyme (200 μL) is added, followed by stirring.The above steps constituted the lysis process.

In the adsorption process, the mixed solution is poured into a spincolumn 8 and centrifuged (number of revolutions 6,000 rpm) for 1 minuteusing a desk centrifuge.

In the washing process, a washing buffer (W2) 11 (500 μL) is poured intothe spin column 8, and centrifugation (number of revolutions 6,000 rpm)is carried out for 1 minute.

In the elution process, an elution buffer (E1) 12 (100 μL) is pouredinto the spin column 8. After standing for 2 minutes at room temperature(25° C.), the nucleic acids are collected by 1 minute of centrifugation(number of revolutions 6,000 rpm). The nucleic acids recovered arequantified, and the purity of the nucleic acids is evaluated.

As a result, highly pure nucleic acids with an A260/A280 ratio of 1.8are obtained. They could be used in the PCR.

The invention provides a method for isolating and purifying nucleicacids with a high recovery yield and shorter time. It thus is excellentin operability and less susceptible to contamination.

The foregoing invention has been described in terms of preferredembodiments. However, those skilled in the art will recognize that manyvariations of such embodiments exist. Such variations are intended to bewithin the scope of the present invention and the appended claims.

1. A method for isolating and purifying nucleic acids, which comprises:providing a mixed solution containing the nucleic acids, salts, and atleast one organic solvent; adsorbing the nucleic acids on an adsorptionsupport; washing the support adsorbed with the nucleic acids with awashing buffer; desorbing the nucleic acids from the support with anelution buffer thereby recovering the nucleic acids, wherein saidorganic solvent includes at least one of ethylene glycol dimethyl ether,ethylene glycol diethyl ether, propylene glycol dimethyl ether,propylene glycol diethyl ether, diethylene glycol dimethyl ether,diethylene glycol diethyl ether, tetrahydrofuran, 1,4-dioxane, propyleneglycol monomethyl ether acetate, ethyl lactate, hydroxyacetone, acetone,and methyl ethyl ketone.
 2. The method for isolating and purifyingnucleic acids as claimed in claim 1, wherein the concentration of theorganic solvent in said mixed solution is not more than 50% by volume.3. The method for isolating and purifying nucleic acids as claimed inclaim 2, wherein the concentration of the organic solvents in said mixedsolution is 5% to 50% by volume.
 4. The method for isolating andpurifying nucleic acids as claimed in claim 1, wherein said mixedsolution contains a surfactant at no more than 50% by volume.
 5. Themethod for isolating and purifying nucleic acids as claimed in claim 4,wherein said mixed solution contains a surfactant at 5% to 50% byvolume.
 6. The method for isolating and purifying nucleic acids asclaimed in claim 1, wherein said mixed solution contains a defoamingagent at 0.2% to 2.5% by volume.
 7. The method for isolating andpurifying nucleic acids as claimed in claim 1, further comprises:providing a column with a bottom; placing the support above the bottom;causing the mixed solution to pass one-way through the support to thebottom by a sucking force.
 8. The method for isolating and purifyingnucleic acids as claimed in claim 7, whereby the causing step, the mixedsolution also passes through the support the other way by an oppositesucking force.
 9. The method for isolating and purifying nucleic acidsas claimed in claim 8, whereby the causing step, the mixed solutionpasses through the support a number of times to enhance adsorptionefficiency.